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. 2017 Apr 17;38(10):1412–1424. doi: 10.1038/aps.2017.35

Figure 5.

Figure 5

The N-terminal negative charge is not required for Notch cleavage by γ-secretase. (A) Sequence alignment of a subset of γ-secretase substrates (human C99 and human Notch family). Shown are TM regions together with flanking N-terminal luminal and C-terminal cytoplasmic regions (C99 [17-61], Notch1 [1721-1767], Notch2 [1666-1710], Notch3 [1629-1675], Notch4 [1432-1481]). Negatively and positively charged residues are highlighted by red and blue letters, respectively. (B) Abolishment of Notch1/2/3 N-terminal negative charge by alanine mutagenesis. (C) Cartoon illustration of C99, C99ΔN, and the C99 hybrid protein, in which the N-terminus is replaced by that of Notch4 [1432-1447] (left). Cleavage efficiencies of these constructs measured by Epsilon-Cleavage assay are shown on the right. (D) Defining the minimum substrate N-terminus for Notch1 by γ-secretase Epsilon-Cleavage assay in living cells. Cartoon illustration of the Notch1 N-terminal truncations (left) and the corresponding cleavage efficiencies (right). Error bars=SEM, n=3, P-values (two-tailed Student's t-test versus WT): *P<0.05; **P<0.01, ***P<0.001.